In eukaryotic cells, proteins are often targeted to specfic compartments. This targeting ususally depends on signals within the proteins themselves that are recognized by specific intracellular receptors. One compartment of interest are peroxisomes. Most proteins are targeted to peroxisomes by virtue of sequences found at their carboxyl-termini that approximate the sequence -SerLysLeu. These are recognized by the protein Pex5p. Sequence variants in the proteins that participate in this protein targeting process have been associated with developmental disorders termed peroxisome biogenesis disorders. Through studies of the binding of a series of peptides to wild-type human Pex5p as well as two disease-associated sequence variants, we have developed a model that rationalizes the peroxisome targeting properties of many proteins within the human proteome. This model provides an explanation of the variation in the severity of disease with Pex5p sequence variation. We have now tested this model by synthesizing peptides that correspond to essentially all proteins encoded by the human genome that are targeted to the inside of the peroxisome by -SerLysLeu-like sequences. The results of binding studies confirm almost all aspects of the model. Furthermore, the affinities of the peptides for Pex5p have been compared with expression levels for the corresponding genes in a wide range of tissues suggesting co-evolution of targeting signals and expression levels with the avoidance of high-affinity targeting signals on highly expressed proteins. This analysis makes a number of predictions regarding tissue-specific peroxisome composition that can be tested. In addition, we have expressed the PTS1-binding domains from Pex5p from trypanosomes (where it functions to target proteins to an organelle termed the glycosome) and found that the domain binds PTS1 peptides with somewhat higher affinity and similar, but not identical, sequence preferences than does the human Pex5p domain. Processes such as inflammation involve cytokines that initiate often complex sigmaling pathways inside cells. Cytokines such as tumor necrosis factor alpha bind to receptors expressed on the surfaces of responsive cells. This binding triggers a signal transduction cascade that eventually leads to changes in gene expression including, in many cases, the expression of additional cytokines. These signal transduction cascades involve many protein-protein interactions. In addition, they also involve chemical modification of specific proteins including the addition and removal of phosphoryl groups and the addition and removal of small proteins such as ubiquitin or chains of ubiquitin. In the case of the tumor necrosis factor alpha-initiated signal tranduction pathway, many of the participating proteins include small structural domains organized around bound zinc ions. We have synthesized peptides that correpond to these zinc-binding domains and found that these protein fragments can often participate in specific protein-protein interactions and in protein modification reactions. In particular, we have found that zinc-binding domains from the protein A20 can both affect ubiquitation reactions involving other proteins and can, themselves, be modified by the addition of ubiquitin. These observations set the groundwork for more detailed studies directed toward determining the structural features that are required for participation in these reactions. We have now completed metal-binding studies on peptides corresponding to all seven zinc-binding domains in A20 and are continuing to test a variety of assays for examining peptide-ubiquitin interactions. Zinc finger proteins typified by Xenopus transcription factor IIIA typically bind double-stranded DNA in a sequence-specific fashion. The determination of DNA-binding affinity and specificity requires a robust binding assay. We have adapted a fluorescene anisotropy-based assay for zinc finger proteins and used in to examine the DNA-binding affinity and specificity as well as the salt-dependence of these parameters for a set of designed zinc finger proteins. This assay is applicable for studies of both naturally occurring and designed zinc finger proteins.